HMGB1, TGF-β and NF-κB are associated with chronic allograft nephropathy

Abstract

The present study aimed to investigate the association between high mobility group protein B1 (HMGB1), transforming growth factor-β1 (TGF-β1), nuclear factor-κB (NF-κB) and chronic allograft nephropathy (CAN) and to identify the clinical significance of HMGB1, TGF-β1, NF-κB on patients with CAN. Between September 2012 and November 2014, 27 patients with CAN diagnosed by biopsy were enrolled in the present study and a further 30 patients that underwent nephrectomy following trauma were selected as the control group. Immunohistochemical staining with HMGB1, TGF-β1 and NF-κB expression in the renal tissues, and western blot analysis were used to measure the relative expression of HMGB1, TGF-β1 and NF-κB. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to estimate the relative expression of HMGB1, TGF-β1 and NF-κB mRNA. Statistical analysis was used to calculate the association between HMGB1, TGF-β1 and NF-κB expression and CAN grade. Immunohistochemical staining demonstrated that HMGB1, TGF-β1 and NF-κB had markedly positive expression rates in renal tubular epithelial cell cytoplasm and membranes in CAN renal tissues, and the positive rates of HMGB1, TGF-β1 and NF-κB increased with the aggravation of CAN pathological grade (I, II and III). The results of western blot analysis indicated that the expression levels of HMGB1, TGF-β1 and NF-κB were significantly higher in the CAN group, compared with the normal group (P<0.05), and the expression levels increased with the progression of CAN grade. A positive association among HMGB1, TGF-β1 and NF-κB expression was identified. RT-qPCR analysis demonstrated that the expression of HMGB1, TGF-β1 and NF-κB mRNA in the CAN group was significantly higher than in the normal group (P<0.05), and the relative expression level of HMGB1, TGF-β1 and NF-κB mRNA not only increased with the aggravation of CAN grade, but was also positively associated with the expression of HMGB1, TGF-β1 and NF-κB, respectively. The abnormal expression of HMGB1, TGF-β1 and NF-κB is therefore, an important manifestation of CAN and the expression of HMGB1, TGF-β1 and NF-κB mRNA in the renal tissues are significantly associated with CAN pathological progression. HMGB1, TGF-β1 and NF-κB may form a signaling pathway that leads to the occurrence of CAN, which induces renal interstitial fibrosis.

Keywords: chronic allograft nephropathy; high mobility group protein B1; kidney transplant; nuclear factor-κB; transforming growth factor-β1.

Figure 1.

Light microscopic observation of (A) tubular atrophy, interstitial fibrosis (arrow) and (B) glomerulosclerosis (arrow). Cytoplasm was stained as red, nucleus was stained as blue. Magnification in (A), ×100; in (B), ×200.

Figure 2.

Electron microscopy observation of (A) a normal glomerulus and (B) transplant glomerulopathy with a well-developed basement membrane along the entire capillary circumference, mesangial expansion, and accumulation of subendothelial deposit. Magnification, ×7,000.

Figure 3.

Expression of (A) HMGB1, (B) TGF-β1 and (C) NF-κB in the renal tissue of patients in the CAN group. Protein was stained as brown. Magnification, ×100. HMGB1, high mobility group protein B1; TGF-mo transforming growth factor-B1 NF-κB, nuclear factor-κB; CAN, chronic allograft nephropathy.

Figure 4.

Western blot analysis of HMGB1, TGF-β1 and NF-κB. (A) Immunobloting of HMGB1, TGF-β1 and NF-κB proteins, with β-actin as the reference gene. Expression levels of (B) HMGB1 (C) TGF-β1 and (D) NF-κB in renal tissues. *P<0.05 vs. control; ^#P<0.05 vs. CAN grade I; ^&P<0.05 vs. CAN grade II. HMGB1, high mobility group protein B1; TGF-β1, transforming growth factor-β1; NF-κB, nuclear factor-κB; CAN, chronic allograft nephropathy.

Figure 5.

Reverse transcription-quantitative polymerase chain reaction analysis of (A) HMGB1 (B) TGF-β1 and (C) NF-κB mRNA expression levels in renal tissues. *P<0.05 vs. control; ^#P<0.05 vs. CAN grade I; ^&P<0.05 vs. CAN grade II. HMGB1, high mobility group protein B1; TGF-β1, transforming growth factor-β1; NF-κB, nuclear factor-κB; CAN, chronic allograft nephropathy.